Mold culture



y' March 31, 1942. Q B AYRES ET AL MOLD CULTURE Filed June 1e, 1939 /v/V of dqaezz'o/L Patented Mar. 31, 1942 NITED STATES ATENT- ortica Niedercorn,

Riverside, Conn., American Cyanamidv Company, N. EL, a corporation of Maine assignors to New York,

Application .l'une 16, 1939, Serial No. 279,473

1 Claim.

The invention relates to the controlled digestion of protein materials and more particularly to the bating of hides and skins by subjecting them to the action of tryptic enzymes of Penicillium roqueforti.

It has been discovered that Penicillium roqueforti produces enzymes which have suicient proteolytic activity under neutral, acid and alkaline conditions to render them commercially attractive in the bating of hides and in similar processes involving the selective digestion of proteins.

It is an object of the present invention to provide these enzymes in the form of a dried proteolyticculture which is suitable for use as a bate. It is a further object to provide a method for the bating of hides and skins using these enzymes as bates, preferably in conjunction with a deliming agent such as ammonium sulfate.

The invention includes both the production of these enzymes from Penicillz'um roqueforti by inoculation of a suitable nutrient medium and cultivation of the inoculated medium and the provision of the enzymes and nutrient medium or carrier in dried form, and also the selective digestion of proteins and the bating of hides and skins by subjecting the materials to the action of these proteolytic enzymes.

As illustrative of the proteolytic activity in alkaline solution of these enzymes produced by Penicillium roqueforti there is shown in the single gure of the accompanying drawing a curve, the ordinates of which are given in terms of standard alkali and the abscissae in terms of pH values. The curve was constructed from values obtained in digesting a casein substrate (sodium caseinate) with a culture of these enzymes at increasing where the alkalinity reaches a pH value of about 9,5.

The preparation of these enzymes is as follows: A suitable nutrient medium in the granular or solid discrete particle condition, such as bran, moistened with an equal weight of water, is inoculated with a culture of Penicillium roquefortz and the inoculated moist bran spread out in thin layers on trays. The inoculated bran is then incubated in an oven maintained at a temperature of about 30 C. and preferably at not higher than this temperature, and at a humidity inthe oven such that the atmosphere therein is saturated but does not contain sufficient moisture to cause deposition of the same onto the bran. The inoculated bran is maintained in the oven until sporulation occurs. After incubation for the optimum period the bran culture may be thoroughly mixed -with 0.2% of cresylic acid in solution, if desired, to improve the wetting out of the bran when used in solution. The culture is then dried at a temperature below 45 C., e. g., 40 C., and may be used as such for bating, or an ammonium salt, e. g.,rammonium sulfate, may be incorporated with the moist mass and the mixture then dried. The bran used for thel culture may or may not be sterilized before the inoculation and likewise the culture may or may not be sterilized, although sterilization of the culture does not appear necessary at the present time;l

The enzymemay be liberated from the dried culture by elution with dilute solutions of Various salts, such as ammonium chloride, ammonium sulfate, sodium chloride, sodium sulfate, etc., and

alkalinities of the sodium caseinate solution.

The undigested casein was precipitated with a standardized solutionof sulfuric acid and sodium sulfate and the filtrate therefrom, containing the digested casein, titrated with 0.1 N-alkali. The tests were conducted by a modification of the procedure of Volhard and Loehlein` for the determinatlon of proteolytic activity of enzymes described in Praktikum der Physiologischen Chemie, part l, pages 258-259, by Peter Rona, 2d ed., Julius Springer, Berlin, 1931. The curve which was plottedv for a pH range of 7-10, slopes off sharply at first and then gradually from the point of neutrality until a pI-I of about 9.5 is reached whereupon it again slopes ofi sharply downward toa pH of about 10. By inspection it will be seen from this curve that the enzymes of the present invention manifest a uniformly good rate of activity in alkalies at least up the eluted enzymes -used in other fields as digestants.

The proteolytic enzymes may be applied to the bating of hides and skins in the form of the combined enzymes and nutrient medium or carrier in any manner now practiced in the art for the application of other tryptic bates. One method now in use involveswashing the hides from the dehairing step, and adding an ammonium salt, such as ammonium sulfate, to the water containing the hides in order to lower their pH Whichvis generally Very high due to the strongly alkaline conditions under which dehairing takes place. Before adding the ammonium salt the hides may be treated for removing lime blast by the addition of a suitable acid, such as hydrochloric acid, to the Water bath containing the hides. After the pI-I of the bath has been suitably adjusted, the enzyme bate is added thereto in an amount determined by the enzyme unit strength to the point of the bate, the kind of hide or skin to be bated,

the extent of bating desired, the 'length of the beting time and the temperature of the bating bath.

For the purpose of illustration, there is de@ scribed in the following example a method for beting of hides with the enzymes of Penicillium foquefort. A

Example Wash a 500 lb. pack oflimed kips from the dehairing bath with water for 15 minutes at 70' F. Heat the washed pack in water to 95 l". and add 5 lbs. of hydrochloric acid thereto, the bath showing red to methyl orange. After three minutes add lime to the bath until it is slightly pink to phenolphthalein. To the bath then add 31/2 lbs. of ammonium sulfate' and about 1% tryptic enzyme of high activity is desired, such as in desizing and dezumming textiles, paper sizing, tenderizing of meat, stripping of gelatin from photographic films and plates.` manufacture of peptones, chewing gum, glue, foods, drugs and biological products or in any field where by their use a protein or a protein degradation product can be reduced tc a lower molecular size of increased solubility. Y

It will `be understood that the above description is intended as illustrative and not as limiting of the invention, the scope of which is deilned by the following claim.

What we claim is:

A dried culture of uniformly high proteolytic activity in the pH range of 7 to about 9.5, said culture consisting essentially of lenzymes of Peuicillium roquejorti on a bran carrier and being the product ot inocculating bran as the nutrient medium with the mold Penicillium roqueforti, incubating the culture under conditions of heat and moisture whereby the bran is impregnated with enzymes of proteolytic activity, and drying the incubated culture.

GILBERT B. AYREs. JOSEPH G. NIEDERCORN. 

